CA2300477A1 - Amplification of nucleic acids - Google Patents

Abstract

The present invention relates to a method of amplifying a nucleic acid sequence present in a first strand of a double stranded nucleic acid molecule wherein said molecule incorporates an unmodified recognition site for a restriction enzyme capable of cutting the first strand at the 5' end of the sequence therein to be amplified and leaving the 3'-end region of the second strand projecting beyond the cut site in the first strand. The method further comprises treating said molecule with said enzyme in the presence of a strand displacing polymerase and unmodified nucleotides for incorporation in an extending nucleic acid strand such that there is or becomes hybridised to said 3'-end region of the second strand a primer sequence complementary thereto whereby said primer sequence is extended in the 5' to 3' direction using the second strand as a template to regenerate the restriction endonuclease cut site and displace the sequence to be amplified.

Description

4.FEH.2000 12:46 MARKS & CLERK MiC 0161 2S6 5846 M0.807 P.6i5S

AMPLIF1C'ATTON OF NI1C'I~FI A 1DS
The present invention relates to the amplification of nucleic acids, i.e.
procedures for producing copies of nucleic acid sequences.
As used herein the term "nucleic acid" includes protein nucleic acid (PNA) (i.e. nucleic acids in which the bass are linked by a polypeptide baclwbone) as well as nucleic acids (e_g. DNA and RNA) having a sugar phosphate backbone.
Various nucleic acid amplification techniques are already known, c.g. the Polymerase Chain Reaction (PCR). I-Iowevor many of these techniques (including PCR) suffer from the disadvantage that vwious cycles of heating and cooling are required for each amplification reaction. Thus, in a typical amplification reaction, the sequence (in single stranded form) to be amplilaed is treated with an oligonucleotide capable of hybridising to the sequence at a particular location thereof, the treatment being effected at a temperature (and under other conditions, e.g. buffers etc_) at which the hybridisation will occur. In the next step (which may or may not be effected at the same temperature) a polymerase enzyme is used to extend the oligonucleotide primer (using the original sequence as a template) to produce a strand which is complementary to the original strand and which is hybridised thereto.
Subsequently the reaction mixture must be heated to denature the complementary strands and then cooled so that the above described procedure (i_e. primer hybridisation, extension, denaturing) play be repeated.
An alternative amplification procedure known as Strand Displacement Amplification has previously been proposed. This procedure may be effected isothermally but does rtquire the constriction of a double stranded nucleic acid molecule incorporating herni-modified restriction site, more particularly a site modified {in one strand) by the incorporation of a thiolyated adenine. The SDA
reaction must be conducted in the presence of a chemically modified base to ensure 4.FEH.2000 12~46 MRRKS & CLERK MiC 0161 236 5846 N0.807 P.'7i53 WO 99109:11 PCT/GB98/02427 regeneration of the hemi-modified restrittion site. This modified base does however become incorporated in the copy strands produced in the reaction and this is a restriction imposed on the procedure.
According to the present invention there is provided a method of amplifying a nucleic acid sequence present in a first strand of a double stranded nucleic acid molecule comprised of complementary first and second strands wherein said molecule incorporates an unmodifted recognition site for a restriction enzyme capable of cutting the first strand at the 5' end of' the sequence therein to be amplified and leaving the 3'-end region of the second strand projecting beyond the cut site in the first strand, and said method comprises treating said nucleic acid molecule with said enzyme in the presence of a strand displacing polymerise and unmodified nucleotides for incorporation in an extending nucleic acid strand under conditions such that there is or becomes hybridised to said 3'-end region of the second strand a primer sequence complementary thereto whereby said primer sequence is extended in the 5' to 3' direction using the second strand as a template to re-generate the restriction endonuclease cut site and displace the sequence to be amplified.
By unmodified recognition site we mean that the site consists of unmodified A, G, T and/or C bases.
It will be appreciated that the steps of cutting and extension may be repeated as often as necessary to generate the desired amplification.
An impoxtant feature of the invention is that the restrictiorx enzyme is capable of providing a 3'-end region of the second sttartd which projects beyond the cut site in the first straltd. The nucleic acid molecule andlor the natuxe of the restriction enzyme 4.FEH.2000 12:46 MARKS & CLERK MiC 0161 236 5846 N0.807 P.8i53 W U 99/092 t 1 PCTlGB98/02427 may be such that only the first strand is cut (i.e. nicked). Alternatively both strands n~.ay be cut. In either case, the cut in the first strand generates a fragment (a 3'-upstrea,m fragment) on the 3' side of the cut in that strand. This fragment may, in ceuain cases, act as said primer sequence (provided that it remains hybridised to the 3'-end region of the second strand) Alternatively, depending on the length of the fragment and/or the reaction conditions, the fragment may be cleaved from the end region of the second strand to generate a 3'-overhang. It is therefore usually preferred that an olig,onucleotide primer (also referred to herein as the FP primer) capable of hybridising to the ;'-end region is additionally incorporated in the reaction to improve the probability of there being a primer sequence hybridised to the 3'-end region of the second strand for effecting the extensionldisplaeement reaction. It will be appreciated that the FP primer incorporates all or part of the overhanging sequence produced by the enzyme digestion outlined above.
The manner in which amplification proceeds. to effect amplification is described in more detail below but, in brief, the primer sequence is extended in the 5' to 3' direction to displace the sequence to be amplified whilst creating a fwther copy of that sequEnce (hybridised to the template strand) and regenerating the restriction site. The processes of cutting the double stranded molecule and extension/displacement are repeated to provide for increasing quantities (i.e.
ampli$cation) of the sequence to be amplified.
It is preferred that the nucleic acid molecule incorporates two restriction sites of the type described, one each side of the sequence to be amplified. These restriction sites are ideally the same as each other (so that only one restriction enzyme is ' required) but may be different. The provision of two restriction sites as described allours for extension/displacement reactions to proceed in opposite directions from either end of the molecule. By providing an excess of FP primers in the reactant mix, these primers may hybridise to the 3'.overhan~gs, produced by the digestion with the restriction enzyme, allowing production of double stranded molecules incorporating a 4.FEH.2000 12:46 MRRKS & CLERK MiC 0161 236 5846 M0.807 P.9i53 single restriction site. These double stranded molecules participate in further amplification reactions as described more fully below. As desczibed below, such , further reactions produce nucleic acid strands wltich are not able to bind to the FP
primers. In an adv~tageous development of the invention, the reactant mix includes at least one further type of primer (referred to herein as ISOS prirners) incorporating the FP sequence and beitlg capable of hybridising to the nucleic acid strands which are themselves not able to bind to the FP primers r se, The ISOS primers result in generation of further double stranded molecules incorporating a restriction site, such molecules being able to participate in amplification reactions. This channelling of otherwise non-hybridisable single strands back into the amplif ration process leads to an exponential accumulation of product. Where both ISO-S and FP primexs are used, the former will generally be employed at a much lower concentration than the latter, generally at least 10 fold less ISO-S primer than FP primer. The ISO-S primers generally are used at a concentration of between 1 fm,ol/~1 to 50 pmol/pl and the FP
primers are generally used at a concentration of between 10 fmol/p.l to 500 pmol/pl.
It should be noted that, under conditions in which the cleavage products of restriction enzyme digestion do not become separated prior to the action of the DNA
polymerase, the exponential amplification reaction may be performed by the 1SOS primers in the absence of FP primers.
The method of the invention may be a solution phase reaction, and must occur under such conditions (of salt concentration, pH, nucleotide concentration etc,) that both the restriction digestion and polymerise extension reactions can occur, though not necessarily simulta~~eously. Preferred conditions for the method of the invention to be carried out are in a New England Thermopolymerase buffer at pH 8.8, in the presence of 10-20 mM magnesium ions, and a nucleotide final concentation of 0.1 -1.0 mM.
Theoretically, the target nucleic acid for the amplification may be present in very small amounts, and hence the amplification will find utility in such areas as 4.FEH.2000 12:47 MRRKS & CLERK MiC 0161 236 5846 N0.807 P.10i53 WO 99/09211 PCTICB98/02$27 clinical diagnostics, to detect either the presence or absence of a sequence or, at a more discriminatory level the occutTence of variations of an underlying sequence.
Both DNA and RNA might be used as the target for amplification, the later requiring initial reverse transcription to a DNA intennediate.
The restriction enzyme may for example be TspRT but any other enzyme producing the required 3'-end region in the second strand may be used.
The strand displacing polymerase may for example be 9°N polymerase (ex-New England Biolabs), IClenow (exo-) polymerase, Bst polymerase, Vent (exo-) polymerase, or Deep Vent (exo-) polymerase.
The temperatures) at which the reaction is effected is/are dependent upon the combination of restriction endonuclease and displacing polymerase used in the reaction. Under terrain conditions, the restriction endonuclease will effect cutting at the temperature at which the displacing polymerase twill effect a copying reaction.
Under such conditions, the reaction of the invention may be effected isothermally. Ii is however also within the scope of the invention that different temperatures are required for cutting and copying so that a two-step thermal cycling reaction may be envisaged. Thermal cycling in this case is not that cycling which is required to perform PCR. In PCR, a strand separation step is an absolute requirement of each cycle of the technique and is usually performed by heating the sample to 95-98°C. No such cyclic strand separation is requited in the method of the invention where cycling is used to allow primer annealing or to move between the temperature optima of the enzymes used.
The invention will be further described by way of example only with reference to the accompanying drawings, in which Fig. 1 illustrates the recognition site for the restriction enzyme TspRI;

4.FEB.2000 12~47 MARKS & CLERK MiC 0161 236 5846 N0.807 P.11i53 WO 99/09Z1I ~'CT/GS98/02d27 Fig. 2 schematically illustratzs a DNA molecule for amplifcation in accordance with the method of the invernion;
Fig. 3 schematically illustrates one embodiiment of the method;
Fig. 4 schematically illustrates a further embodiment of the method;
Figs. ~.-11 illustrate the results of the Examples.
Referring to the drawings, fig. 1 illustrates the restriction site of the enzyme T.spRI. This recognition sequence comprises five base pairs as shown and the enzynrae cuts two bases either side of the recognition sequence to produce a 9-base 3' overhang.
REferenee is now made to Fig. 2 which illustrates a double stranded target DNA rnolcculz 1 containing an internal sequence X (flanked by sequences Y and Y') to be amplified by the procedure described more fully below with reference to Fig. 3.
l~l~ith reference to these figures, the following convention will be used when referring to the composition of fragments. Sequences which are complementary are denoted by the samE letters (or sequence of letters) but with one of tl~e two complementary sequences additionally being denoted by the "prime" suffix ('). Thus X' is the complementary sequence to X.
When a molecule is composed of more than ogle segment it will be annotated from the 5' end of the molecule {thus the notation YXY' would be used to denote the upper strand of the molecule in Fig. 2). When a double stranded molecule is referred to the upper and lower sequences are separated by a slash (~ with both annotated from their 5' ends. Thus the entire molecule shown in Fig. Z can be denoted yXY'/YX'Y'.
The molecule shown in Fig_ 2 has a TspRI restriction site towards each end thereof flanking the sequence XlX' with each site being spaced from its respective end of the molecule by a short (8 bp) sequence to distance the restriction site away from the 5' end of the molecule, These 8 hp sequences, which axe identical at each end of the molecule, havE been included because certain restriction enzymes are unable to 4.FEH.2000 12:47 MRRKS & CLERK MiC 0161 236 5846 N0.807 P.12i53 WO 99/09211 PCT/GB98/024z7 cleave near the ends of DNA. It has not been reported whether TspRI behaves in this fashion so that this sequence may or may not be necessary. Likewise, the optimt>rn length of this sequence has not been reported and so this spacer may contain mom or less than 8 bases.
It will be appreciated that the 1? base sequences at the 5' ends of the two strands ( A, and B) of the molecule 1 are identical and are referenced in Fig.
Z as Y and I'..
In order ~o effect the amplification reaction, the molecule 1 is treated with the enzyme TspfiI in the presence of a strand displacing polymerise, a primer FP1 having the aforementioned sequence Y, and the four nucleotides in a buffer such as conventionally used in amplification reactions, A manner in which the reaction may proceed is illustrated in Fig. 3 in which the dashed vertical line is included purely to illustrate the relationship between the various steps of the process.
Initially, the molecule 1 is cleaved at both ends by TspRI so as to produce a ''cleaved" molecule having 3' overhangs (each 9 bases) as illustrated as the result of step (a) in Fig. 3_ In the next step, the primer FP1 (shown in bold for the purposes of clarity) hybridises tv the "cleaved" molecule to produce the construct illusi~rated as the product of step (b) of Fig. 3. It should at this point be appreciated that exactly the same construct would be obtained if the short cleavage fragments of sequence Y
nicked by the TspRI did not become denatured or if such denaturation did occur they became rehybridised.
In the next step, the strand displacing polymerise begins to extend each primer FP 1 so that (with reference to tlae primer FP 1 shown at the left hand end of Fig_ 3) the primer FP1 is extended by copying strand X' as a template arid simultaneously 4.FEH.2000 12:48 MRRKS & CLERK MiC 0161 236 5846 N0.807 P.13i53 1~V0 99/09211 PCT/Cr898/024:7 g displaces the remainder of strand X. Similarly the other FP1 primer is extended by copying of strand X and displaces the remainder of strand X'. Simultaneously with extension of primers FP1, the 3' ends of the cleaved target molecule are extended using the FP 1 primers as a template regenerating the restriction endonuclease sites, All of these copying reactions art schematically illustrated between steps (cl) and (c2) of hig. 3.
For the purpose of simplicity, Fig. 3 shows the product of the strand displacement polymerisation (i.e. the product of step (e2)) to be two, foreshortened, double stranded copies of the original molecule (YX/X'Y' and XY'/YX'). It can be seen that each of these copies lacks one terminus of the original including the TspRI
site at the end of the molecule.
As depicted, the next steps (steps (d) and (e)) involve cleavage of the molecules with TspRI and hybridisation of printers FP 1 to the 3' overhangs produced.
Once these primers are hybridised to the respective strands, strand displacing primer extension can occur as shown (steps (fl) and (f2)). The products of this extension include two foreshortened double su~anded molecules (YX/X'Y' and XY'/YX') essentially identical to the products of steps (cl) and (c2). These molecules are, therefore, available for further cycles of TspRI
cleavage/hybridisation/extension (steps (d), (e) and (fl/f2)). The other products of step f ((fl) and ('i2)) are two, ftuther shortened, single strand sequences (X and X') which contain neither TspRI
restriction site, These sequences are complementary, being copies of each strand of the central region of the original molecule-With the method as thus far described, these single strands X and X' do not participate further in the amplification reaction. Amplification znay however be greatly enhanced by incorporatiilg, in the reaction mix, additional primers ISOI and ISO? (see Fig. 3(g)). ISO1 has a 5' terminal sequence corresponding to Y above whereas the remainder of the primer may be hybridised to the 3' end of sequence X' 4.FEH.2000 12:48 MARKS & CLERK MiC 0161 236 5846 N0.80'7 P.14i53 WO 99/09211 PCTlGB98/02427 (Fi ; 3 (g)), Primer ISOZ also has 5' sequence corresponding to Y and the remainder of the primer may be hybridised to the 3' end of sequence X (Fig. 3 (g)).
Polymerase extension, once again. "fills in" the molecules (steps (hl ) and (h2)) to generate two foreshortened double stranded products. These molecules (YXIX'Y' and XY'/YX') are identical, in sequence terms, to the double stranded products of steps (cl/c2) and (fl/fZ) described earlier, and so, are also able to re-enter further cycles of TspRl cleavage/hybridisation/extension (steps (d), (e) and (fl/f2)) described earlier.
Therefore, to summarise, each original molecule 1 undergoes a reaction resulting in the production of two shortened ptroducts lacking one or other terminal restriction site. Further processing oC these molecules leads to their cyclical regeneration with the additional production of single stranded copies of each strand of the region between the TspRl sites in the original molecule. In the presence of the longer, target sequence specific ISO-primers (ISOI and IS02) these single strands are processed to generate further molecules identical to the earlier shortened double strands. The o~eratl process is depicted in Fig. 3. The reaction thus cycles from (d) to (h) in a geometric manner.
It should be noted that primers ISO1 and IS02 should be at much lower concentration than primer FPl so that in step (b) of Fig. 3 there is a much higher probability of primer FP 1 hybridising to the 3'-overhang than either of ISO1 oz IS02 which could not then be extended to re-generate the restriction site.
For the purposes of simplicity, the description so far has assumed that the reaction occurs in "discrete" steps. Thus for example, with reference to steps (c 1 ) and (cz) it has been assumed that the copying reactions illustrated therein are completed before any further cutting of the molecule. In practice, this may not actually be the case since once the restriction site has been re-created it may be nicked once again before the copying reaction illustrated in steps (el) and (c2) is completed.
This has not however effected the overall result of the process as described above.

4.FEH.2000 12:48 MARKS & CLERK MiC 0161 236 5846 N0.80~ P.15i53 w0 99/09211 PCTlGB98/024Z7 A number of modifications may be made to the process.
For example, the process may be performed using two different sequences PP 1 auld FP2 instead of the single sequence FP1 as described.
Furthermore, under certain conditions, the cleavage site (generated by TspRI~
may not dissociate prior to extension by nucleotide polymerase so that, under such conditions, the amplification reaction will proceed without the need to use primer rPl. This is illustrated in the procedure of l~ig. 4 which, in many respects, is analogous to the procedure shown in Fio. 3, save that.
(i) the printers FP 1 are not used; and (ii) the cutting of double stranded molecules is shown to yield seventeen base pair fragments designated SFI, which do not dissociate from the molecule and which, as will be appreciated from fig. 4, have a priming function in the same manner as primers FP 1.
Iii further modifications of the procedure, alternatively or additionally there may be only one restriction site per molecule or different restriction sites at the opposite ends of the molecule.
Alternatively or additionally the restriction site may be that of a restriction enzyme other than TspRI.
It will be appreciated that, if the sequence to be amplified is present in a single stranded molecule then a double stranded molecule containing the required restriction site may be generated therefrom using knouvn techniques.

4.FEH.2000 12:49 MARKS & CLERK MiC 0161 236 5846 fJ0.807 P.16i53 The invention is illustrated by the following non-limiting Examples.
m le An amplification reaction was effected using the following templates, primers and reaction conditions.
Template 400 by PCR fragment (Isofragment) generated froth pUCl9 using primers ISOSI and ISOS2 below;
Primers: Two sequence-specific primers ISOSi and ISOS2> and one Forcing Primer, ISOFP1 (equivalent to FP1 in Fis. 3) as shown below;
ISOSI (SEQ ID NO. 1): 5'-TAATC TTTGG CAGTG GCTTA CAACG
TCGTG ACTGG GAAAA C
ISOS2 (SEQ ID NO. 2): 5'-TAATC TTTGG CAGTG GCTGA CGGTG
AAAAC CTCTG ACAC T
)CSOFP1 (SEQ ID NO. 3): ~'-TAATC TTTGG CAGTG GC
Reaction Conditions:
20~L New Englarnd Biolabs Buffer 4 10~L Isofragment (SOfmol uL-~) Sp. dNTPs (IOmM each) ' lp.L ISOS2 (4 pmol ~L-~) 2~L ISOS1 (2.2 pmol ~L~~) lp.l ISOFP1 (40pmo1 N.L'~) S~TspRI (5 U itL-~) I p.L Bovine Serum Albumen (NEB 100x concentrated) 4.FEB.2000 12:49 MRRKS & CLERK MiC 0161 236 5846 NO.B07 P.17i53 w0 99!09211 PCT/G~98/02a27 lz 170 yL HZ(l A mix containing the three primers (ISOSI and ISOS? at approx. 20 fmol ~tL-~, and ISOFP1 at approx. 200 fmol ~L-~), deoxyribonucleotides (250 p.molh~
each), and template (lsofragment 2 at ?.5 fn~ol p.L'~) was equilibrated (in a water baih) to 65°C when 25 U TspRI restriction enzyme was added. Tl~e optimum temperature for cleavage of Isofragment 2 by TspRI is 65°C and incubation was continued for 20 minutes to allow restriction to occur (Fig. 3{a)). The mix was transferred to a second water bath at 37°C. A short equilibration period (30s - lm) was then included to allow hybridisation of primers to exposed ssDNA ends of the fragment (Fig.
3(b)).
101,1 of Klenow (exo-) DNA polymerase was added, to extend the hybridisation products, which had accumulated, following restriction. The extension reaction was continued for 5 m at 37°C (Fig. 3(cl/c2)). The mix was transferred to the 65°C water bath to initiate a second restriction cleavage by TspltI (Fig. 3(d)).
Following 20 minutes digestion and 30s - !min hybridisation (Fig. 3(e) and (g)) further Klenow enzyrrte (l0U) was added and extension continued for 5 minutes (Fig 3. (11/2) and (h1/2)). Since TspRI can sulwive incubation at lower temperatures (e.g.
37°C) the initial amount input was sufficient for the entire reaction. In contrast, Klenow (exo-) is destroyed by prolonged incubation at 65°C and so additional enzyme was added to the mix following each incubation at the higher temperature. The cycle of incubation at 65°C, hybridisation, Klenow addition and extension at 37°C
constitute the "cycles"
of amplification. Samples (20 ~tL) were removed at the end of the cycle as necessary.
The reaction in the samples taken was ternninated immediately by the addition of 4p.L
of agarose gel loading dye and samples were held at 4°C anti) all samples were collected. The samples were then analysed by agarose gel electrophoresis in 1%
(wlv) agarose gel, containing 0.5pg mL-~ ethidium bromide, in 1 x TBE buffer.
The results of this procedure are shown in Fig, 5 which demonstrates that amplification was clearly visible In all cases.

4.FEH.2000 12:49 MARKS & CLERK MiC 0161 236 5846 N0.807 P.1Bi53 The above procedure was repeated but using an extension temperature of 39°C
instead of 37°C. A repeat was also conducted using the extension temperature of 39°C but omitting the ISOFPI primer from the reaction mix. The results of these procedures are shown in Fig. 6, Tlie importance of including the primer ISOFP1 was clearly demonstrated by Comparing amplification, at 39°C, in the presence and absence of the primer.
The effect of ISOFP 1 on amplification can be quantified, to an extent, by using the gel scanning software (GELSCAN-UVP Ltd). To estimate peak heights or peak areas for the specific band being amplified (ca. 400 pairs). The results of peak area analysis for the gel photograph of Fia. 6 are illustrated in rig. 7.
These results show that them is considerable amplification of the fragment in the presence of ISOFI'1. There is also a small, but significant, amplification in the absence of ISOFP 1.
xa An amplification reaction was effected using the following template, primers and reaction conditio~ns_ Template Purified I50CIv1V template (144bp) at a concentration of 829fmollp,l (produced by PCR of Cytomegalovirus using ISOCMV001 and ISOCMV002 primers).
CMV FRAGMENT (SEQ ID N0. 4) TTAAGTTACG CACTGAGGAA TGTCAGCTTC CCAGCCTCAA GATCTTCATC
GCGGGGMCT CGGCCTACGA GTACGTGGAC TACCTCTTCA AACGCATGAT
TGACCTCAGC AGTATCTCCA CCGTCGAGAC AGTGGTGACA AGAC-3' Primers 4.FEH.2000 12:49 MRRKS & CLERK MiC 0161 236 5846 N0.807 P.19i53 WO 99/09211 PCT/C1398/D2a27 ISOCMV001 36-mer (SEQ ID N0. 5) S' TTA AGT TAC GCA CTG AGG AAT GTC AGC TTC CCA GCC 3' ISOCMV002 37-mer (SEQ ID NO. 6).
~' (iTC TTG TCA CCA CTG TCT CGA CGG TGG AGA TAC TGC T 3' rPCMV003 17-mer (SEQ 1D NO. 7).

5' 1'TA AGT TAC GCA CTG AG 3' FPCMV004 17-mer (SEQ ID NO. 8).
5' GTC TTG TCA CCA CTG TC 3' FP primers were used xt a concentration of SOpmol/p.l while the longer ISO
primers were used at a concentration of Sptnol/ul.
Reaction Conditions:
The following reactions were set up in 0.2m1 flat-topped thin-walled PCR
tubes and incubated, following enryme addition, at 6~°C (in a Biometra TRIO
Thernioblock). The time points fox incubation were 0, ?0 40 and 80 minutes. At the appropriate time, 6p1 of agarose gel loading dye was added to each reaction mix and samples were placed at 4°C until all time points were completed.
ISOCMV001 primer 2~1 ISOCMV002 primer 2p.1 FPCMV003 primer 2p.1 PPCMV004 primer 2~1 ISOCMV Template 0,5E,,1 SOmM magnesium chloride (GIBCO) 6p.1 x Thermopolymerase buffer (New 3p.I
England Biolabs) 1 OmM dNTP (Pharmacia) 1 ~l 4.FEB.2000 12:50 MRRKS & CLERK MiC 0161 2S6 5846 N0.807 P.20i53 WO 99/09211 PCT/GIi98/024Z7 TspRI restriction enzyme, SU/p.l (New England Biolabs) 2p1 Bst Polymerase, 4U/~1 (New England Biolabs) 0.5p.1 100 x Bovine Serum Albumin (New England Biolabs) (diluted to 1X with Molecular Biology grade water) 9~1 Total Reaction Volume 30p,1 Following incubation twenty microlitres of each sample was analysed by loading on a 10% polyacrylamide, IxTBE (non-denaturing) gel using a Bio-Rad Mini-PROTEAN gel system. The gel was run for approximately 1 hour at 100-150V
and then stained for 20-30 minutes in a solution of 0.5 mg/ml ethidium bromide/1 x TBE {diluted from a stock of lOmg/ml solution from Sisma).
The gel was visualised using a UV transilluminator and photographic system (MWG Biotech). An image of the gel is show in Fig. 8 and demonstrates considerable amplification under the conditions used.
am le 3 Template: 400 by PCR fragment (Isoftagment) senErated from pUCl9 using primers ISOS1 and ISOS2 below;
Primers: Two sequence-specific primers ISOS1 and ISOS2, and one Forcing Pzitner, ISOFP1, as shown below;
ISOS1 (SEQ ID NO. )<): 5'-TAATCTTTGGCAGTGGCTTACAACGTCGTGACTGGGAAAAC
ZSOS2 (SEQ ID NO. 2): 5'-TAATCTTTGGCAGTGGCTGACGGTGAAAACCTCTGACACAT

4.FEH.2000 12:50 MRRKS & CLERK MiC 0161 2H6 5846 N0.807 P.21i5S
WO 99/09211 PCT~GB98/02427 ISOTPi (SBQ ID NO. 3): 5'- TAATCTTTGGCAGTGGC
Reaction Conditions:
20ph New England Biolabs Buffer 4 10~L Isofragment (SOfmo1 ~.L'~) ~L dNTPs { 1 OmM each) 1 pL ISOS2 (4pmo1 ~L'~) 2~L ISOSI (z.2 pmol pL-~) lltL ISOFP1 (40pmo1 ~.L'~) 5~L TspRl (5 U ~L-~) 1 pL Bovine Serum Albumen (NEB I00x concentrated) 170 ~L H~0 A mix containing the tlvee primers (ISOS1 and IS052 at approx. 2afmoi ~L-~, and ISOFP1 at approx. 200 fmol ~tL-~), deoxynucleotides (250 ~mol ~L'~ each), and template {lsofrxgment 2 at 2.5 fmol ~L-~) was equilibrated (in a water bath) to 65°C, when 25 U of TSpRI restriction enzyme was added. Incubation was contixiaed for minutes when the mix was transferred to a second water bath at 34°C.
After a short equilibration period (30s ~ 1 m) l0U of Klenow (exo-) DNA polymerase was added and extension continued for 5 m at 34°C prior to returning the mix to the 65°C water bath to initiate another 'cycle' of amplification. Samples (20 pL) were removed at the end of the cycle as necessary. The reaction in samples taken was terminated immediately by the addition of 4 ~L of agarose loading dye and samples were held at 4°C until all samples were collected, The samples were then analysed by agarose gel electrophoresis in 1% (w/v) agarose gel, containing 0.5pg mL'~ ethidium bromide, in 1 x T$E buffer.
The results of this ampliftcation procedure are shown in the gel photograph of Fig, 9 front which amplification is clearly demonstrated.

4.FEH.2000 12:50 MF1RKS & CLERK MiC 0161 236 5846 N0.807 P.22i53 w0 99/09211 PCT~GB98/024?7 Fig. 10 is a plot showing the accumulation of product (400bp band) based on peak arcs analysis of the Gell3ase/GelBlot analysis package (Ultra Violet Products Limited, Cambridge, UK). The 10 cycle value is not shown.
These results show that there was considerable amplification of the fragment.
le 4 Template: purified 144bp PCR fragment (CMV144) generated from human cytomegalovirus DNA using ISOCMVODI and ISOCMV002-AT primers below (used at a final concentration of 760 fmollp.l), Primers:
ISOCMV001 (36-mer) (SEQ ID NO. S):
5' TTA AGT TAC GCA CTG AGG AAT GTC AGC TTC CCA GCC 3' ISOCMV002-AT (37-mer) (5EQ ID NO. 9):
~' ATA TTG TAA CCA CTG TCT CGA CGG TGG AGA TAC TGC T 3' Both primers were used at a concentration of 5pmol/ul Reagents:
Wheat-germ tItNA (~a l Opglml (SIGMA) Lot 87H4045. Stock at l0mglml 50mM magnesium acetate (SIGMA) Lot No. 77H10581 x Thermopolymerase buffer (New England Biolabs) Lot No. 20 l OmM dNTP (Pharmacia, Polymerisation mix) Lot 7122094021 TspRI restriction enzyme, SUlp.l (New England Biolabs) Lot No. 2 Bst Polymerise, 8U/p.1 (New England Biolabs) Lot No. 13B
Agarose Gel Loading dye, AGL007 Molecular Biology grade water from ELGA water purifier Equipment:

4.FEB.2~00 12:50 MARKS & CLERK MiC 13161 236 5846 N0.807 P.23i53 Biometra 1"RIO Thermoblock Method:
Template (CMV 144) was diluted from the 760 fmol/~.l stock using serial tenfold dilutions. Concentrations of template used in the assay were 760 zmol/1<1 (456000 molecules/ltl), 76 zmolllLl (45600 molecules/lil), 7.6 zmoUul (4560 molecules/hl), 0.76 zmol/ltl (456 molecules/p.l), and 0.076 zmol/~1 (456 moiecules/p,l).
The following reaction was set up in 0.?ml flat-topped thin-walled PCR tubes, using each of the five template concentrations detailed above, and incubated at 5GC

for ? hours.

ISOCMV001 primer 21<1 ISOCMV002 primer 2111 50mM MgAc 511 IOmM dNTP O.S1L1 x Thermo buffer 5k1 TspRI 1.51>,l Bst Polymerase 0.511 template (CMV144) dilutions0.5p.1 MB water up to 2511 7.51 ~'otal Reaction Volume 25111 Fifteen microlitres of sample was analysed by loading on a IO%
polyacrylamidc, IxTBE (non-denaturing) gcl using a Bio-Rad Mini-PROTEAN gel system. The gel was can for approximately 45 mins at 175V and then stained for minutes in a solution of O.Smg/ml ethidium bromide/1 x TBE (diluted from a stock of l0mg/ml solution from Sigma).

4.FEH.2000 12:50 MRRKS & CLERK MiC 0161 236 5846 N0.807 P.24i53 The gel was visualised using a UV transiluminator and photographic system (MWG Biotech), The results of this amplification procedure are shown in the gel photograph of Figure 11 from which amplif canon is clearly demonstrated, In the above Examples, the targets used contained sites for the restriction enryme, TspRI. which were introduced during their production. It will however be appreciated that suitable targets may be generated in a number of ways. Such methods might include, but are not limited to:
1, Restriction digestion of long nucleic acid sequences carrying the target, to generate fragments with at least one known end. lire restriction enzyme may, or may not, be that used for subsequent amplification, however, for exponential amplification the ISO primers must contain 3' sequences homologous to the 3' sequences of the fragment produced. Denaturation of the fragment (e.g. by elevated temperature) similar in structure to X and X' shown in Fig. 3. (F2) and thus the amplification would begin from that point.
Z. Ligation of two synthetic oligonucleotides, using the target molecule as template, in a manner that such ligation generates a product which, when separated from the W rget by denaturation, as above, has a structure YX or YX' or YXY' or YX'Y', using the nomenclature as in Fig. 3. Such molecules would be a target for hybridisation by FP1 or an appropriate ISO primer, and would generate double stranded intermediates in the amplification cycle following polymerise extension.
3. Reverse transcription of an RNA target molecule with a primer containing the target specific sequence flanked by a sequence containing a suitably positioned restriction endonuclease site (ISO primer). Following separation of the RNA
from the extended DNA copy product the addition of a target specific "downstream"
primer (or ISO primer) would initiate the amplification reaction.

4.FEH.2000 12:51 MRRKS & CLERK MiC 0161 236 5846 N0.807 P.25i53 WO 99!0931 L ~C"~'/G~98/024~7 SEQUENCE LISTING
( 1 ) GENER_~.L INFDRMATION
(i) AppLICANT:
(A) VAMP: TEPNEL MEnICAL LIMITED
(H) STRE~T: UNIT e, ST. GEORGE'S COURT, HANOVER HUSINESS
PARK
(C) CITY: ALTRINCFdAM
(D) STA?E; CHESHIRE
(E) COUNTRY: UNITED KINGDOM
(F) POSTAL CDDE (ZIP): WA14 5UA
(ii) TITLE OF INVENTION: AMPLIFICATION OF NUCLEIC ACIDS
(iii) N~mIB~R OF SEaUENCES: 9 (iv) COMPU?;~R READABLE FORM=
(A) MEDIUM TYPE: Floppy disk (H) COMPUTER. I8M PC compatible (C) OPERATING SYSTEM: PC-DOS/M5-DOS
(D) SOFTWARE: Pa~entln Release ~l,p, Version (~1.3p (EPO) (2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CkiARAGTERISTZCS:
(A) LENGTH: 41 base pairs (a) TYPE: nucleic zcid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomicl (ixi) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCB CHARACTERISTICS:
(A) L>rNGTii: 40 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY. liaeaz (ii) MOLECULE TYPE: DNA (genamic) (iii) HYPOTHExICAL: NO
(xi) SEQLtENCE DESCRIP?ION: SEQ ID NO: 2:
TAATCTTTGG CAGTGGCTGA CGGTGAAAAC C?CTGACACT 40 (2) INFORMATION FOR SEQ ID NO: 3:

4.FEH.2000 12:51 MARKS & CLERK MiC 0161 236 5846 N0.807 P.26i53 WO 9910921 i PCTIGB98/U2d27 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDGDNESS; single (D) TOPOLOGY: linear (ii) MOLECULE TYPE; DNA (genomic) (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TAATCTTTGG CAGTGGC
(z) INFORMATION FOR SEQ TD N0; 4:
(i) SEQUENCE CHARACTERISTICS:
(AI LENGTH: 19A base pairs (H) TYPE: nucleic acid (C) STRAND&DNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Igenomic) (111) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

12) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (9) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYpE: DNA (genomic) (iii) HYPOTi;ETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TTAAGTTACG CACTCZ~GGAA TGTCAGCTTC CCAGCC 36 I2) INFORMATION FOR 5EQ ID NO: 6:
( i ) 9BQtJENCE CHARACTL~RISTICS ;
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRAND&DNESS: single 4.FEH.2000 12:51 MARKS & CLERK MiC 0161 236 5846 N0.807 P.2'7i53 w0 99/09211 PCT/Gg98/0z427 (D) TOPOLOGY: linear (iiI MOLECULE TYPE: DNA (genomie) (iii) HYPOTHETZC?.L: NO
(xi) SEQUENCE DESCRIPTION: SEQ Ib NO: 5;

(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l~ base pairs (S) TYPE. nucleic acid (C) ST)2ANDEDNESS: single (D1 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
TTAAGTTACG C~CTG.~G 17 (2) INFORMATION FOR SEQ ID NO: e:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7.7 base pairs (8) TYPE. nucleic acid (C1 STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ge~tomic) (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: A:
GTCTTGTCAC CACTGTC
(Z) INFORMATION FOR SEQ ID N0: 9:
(i) SEQUENCE CHARACTERISTTCS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C1 S'~RANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ~'YpE: DNA (genomic) (iiil HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATATTGTAAC CACTGTCTCG ACGGTGGAGA TACTGCT

Claims (9)

1. A method of amplifying a nucleic acid sequence present in a first strand of a double stranded nucleic acid molecule comprised of complementary first and second strands wherein said molecule incorporates an unmodified recognition site for a restriction enzyme capable of cutting the first strand at the 5' end of the sequence therein to be amplified and leaving the 3'-end region of the second strand projecting beyond the cut site in the first strand, and said method comprises treating said nucleic acid molecule with said enzyme in the presence of a strand. displacing polymerase and unmodified nucleotides for incorporation in an extending nucleic acid strand under conditions such that them is or becomes hybridised to said 3'-end region of the second strand a primer sequence complementary thereto whereby said primer sequence is extended in the 5' to 3' direction using the second strand as a template to re-generate the restriction endonuclease cut site and displace the sequence to be amplified.

2. A method as claimed in claim 1 wherein the nucleic acid molecule incorporates two of said restriction sites, ore each side of the sequence to be amplified.

3. A method as claimed in claim 1 or 2 wherein the or each restriction site is a TspRI site.

4. A method as claimed in any one of claims 1 to 3 wherein the reaction mixture additional incorporates an excess of oligonucleotide primers (FP) incorporating the restriction sequence and capable of hybridising to the 3' end of the displaced sequence whereby a double stranded molecule incorporating the restriction site is generated and the latter double stranded molecule is capable of participating in further cutting and extension/displacement reactions to generate strands which do not incorporate the restriction sequence.

5. A method as claimed in any one of claims 1 to 4 wherein the reaction mixture additionally incorporates oligonucleotide primers (ISOS) incorporating the restriction sequence and being capable of hybridising to said strands which do not incorporate the restriction sequence.

6. A method as claimed in any one of claims 1 to 5 wherein the nucleic acid is DNA.

7. A method as claimed in any one of claims 1 to 6 wherein the strand displacing polymerase is 9°N polymerase, Klenow (exo-) polymerase, Bst polymerase, Vent (exo-) polymerase, or Deep Vent (exo-) polymerase.

8. A method as claimed in any one of claims 1 to 7 effected isothermally.

9. A method as claimed 1n any one of claims 1 to 8 wherein the DNA molecule is generated in situ by reaction of s precursor nucleic acid molecule containing the sequence to be amplified with at least one primer capable of generating the restriction site.